The release of cytochrome c from mitochondria is a critical step in the major pathway of caspase activation and apoptosis affecting mammalian cells. However, mitochondrial outer membrane permeabilization (MOMP) appears to commit cells to die regardless of whether or not caspases are activated downstream of MOMP. Although this caspase-independent death (CICD) was first described almost 10 years ago, and has been well characterized, there is little consensus as to how it occurs. This proposal is based on our studies that identify conditions under which cells can be protected from CICD but not apoptosis. Using an unbiased screen, we identified GAPDH as being capable of rescuing cells subjected to conditions for CICD, and further, found that this protection acts downstream of MOMP. That is, cells release cytochrome c and subsequently heal their mitochondria and sustain clonogenic growth. Additional studies led us to another protein capable of protecting mammalian cells from CICD: the C. elegans Bcl-2 family member CED9, which does not protect mammalian cells from apoptosis, but effectively preserved clonogenic survival in the presence of caspase inhibitors. The protection from CICD afforded by GAPDH or CED9 involves elements of the autophagic survival pathway, as well as aspects of mitochondrial dynamics. The current proposal seeks to dissect how CICD occurs, and how protection from CICD works. Our specific goals are to answer the following questions: 1. How do cells survive the process that normally leads to CICD? Here, we will investigate the cellular changes that occur during CICD, especially those relating to mitochondrial number and function, and how these are affected by GAPDH and CED9 to promote cell survival. Studies in this aim will identify key events in this process and place them into the context of physiologically relevant events in cell death and survival. 2. How does autophagy contribute to protection from CICD? Autophagy plays critical roles in survival of stressed cells. We will explore how GAPDH enhances autophagy to remove damaged mitochondria, and how this contributes to protection from CICD. 3. How do mitochondrial dynamics contribute to protection from CICD? Mitochondrial fusion is an essential cellular function, and is impaired following MOMP. Both GAPDH and CED9 prevent mitochondrial fragmentation following MOMP, and therefore the role of mitochondrial dynamics in restoring a full mitochondrial cohort and protection from CICD will be explored. Overall, we will test the idea that cells can survive MOMP if a) caspases are not activated, b) metabolic energy is sustained, c) damaged mitochondria are removed by autophagy, and d) a seed pool of intact mitochondria that have either not undergone MOMP or have repaired their outer membranes can repopulate the cell via mitochondrial dynamics. Our studies employ a number of novel approaches and concepts, all of which are directed at answering these questions. Successful completion of the proposed work will reveal fundamental new processes in the control of cell life and death.